*2.1. Leishmania Parasites*

*Leishmania* parasites cycle between the motile promastigote form in the gut of the sand-fly vector and the intracellular amastigote stage within the macrophages and other types of mononuclear phagocytic cells of the mammalian host. In this way, when invading macrophages, *Leishmania* promastigotes block the phagosome maturation process and create an environment that is propitious to amastigote differentiation. Subsequent divisions and later infection of other mononuclear phagocytic cells, as well as different tissues, leads to the setup and progression of the clinical manifestations related to these diseases [26]. Traditionally, compounds have been evaluated by means of cell-free assays using axenic promastigotes and amastigotes, which allow high-throughput screening and high reproducibility, while relying on a limited number or parasites per evaluation. However, these two parasite forms present several important caveats that can lead to the selection of false candidates. On the one hand, promastigotes are not the mammalian form, and they show significant differences in their metabolic profile when compared to intracellular amastigotes. Moreover, their growth and sensitivity are influenced by different parameters, such as cell culture density, medium composition, and compound mode of action (MoA), among others, so care must be taken in interpreting the data [27]. While closer to the mammalian form, axenic amastigotes retain some promastigote traits, leading to a lack of correlation between axenic forms screenings and intracellular amastigote assays, which increases the false-positive rate of hit discovery when using this artificial form [28]. Consequently, models using the intracellular amastigote infecting mammalian host cells remain the gold standard in the determination of drug sensitivity. These models have great advantages such as the direct evaluation of drug penetration in the host cell, as well as drug activity in the phagolysosome milieu, among others [29,30]. Moreover, intracellular amastigotes are generally more sensitive than promastigotes against most of the drugs currently used in clinic, such as antimony or miltefosine [31,32], which could be a consequence of genes differentially regulated in the two developmental stages of the parasite [31,33,34]. The activity of candidate compounds against intracellular amastigotes is determined by microscopic automatic/manual counting of infected macrophages and the number of parasites per macrophage (parasitic index) or by spectrophotometric (e.g., optical density or staining) and fluorometric methods. These latter include the automated detection and quantification of genetically engineered amastigotes that express fluorescent and bioluminescent reporters, which enables faster read-outs and higher throughput [35]. Nonetheless, determination of the cidal and static effects of candidate compounds against intracellular forms can be very challenging, in part because of the slow

replication rate of amastigotes when compared to promastigotes [36–38]. Moreover, this determination could be biased by many confounding factors that can reduce lab-to-lab reproducibility and lead to false hit discoveries. These factors could include macrophage infection rate, incomplete amastigogenesis, impact of distinct culture media, as well as the intrinsic pathogenicity of the strain selected for the assay [39–41].

Despite these potential limitations, in vitro amastigote assays (infecting THP-1 and primary mouse macrophages (PMM cells)) have led to the discovery and optimization of a novel series of amino-pyrazole ureas with potent antileishmanial activity [42]. Likewise, more recently, Van den Kerkhof et al. (2018) evaluated three antileishmanial leads series (nitroimidazoles, oxaboroles and aminopyrazoles) using intracellular *L. donovani* and *L. infantum* amastigotes infecting PMM, and showed a good in vitro to in vivo correspondence, with high efficacy and negligible side effects in vivo [43]. Tunes et al. (2020) found that gold(I)-derived complexes were very active against *L. infantum* and *L. braziliensis* intracellular amastigotes infecting THP-1 cells, including antimony-resistant strains (SbR), and they were potent inhibitors of trypanothione reductase. Moreover, two of these complexes presented very favorable pharmacokinetic and safety profiles in vivo after oral administration [44]. In the search of more robust, scalable, and reproducible models, Melby′ s team developed an ex vivo splenic explant assay that allows the identification of new compounds active against *Leishmania* within the pathophysiologic environment [45,46]. In this way, they recovered the spleens of hamsters infected with a luciferase-transfected *L. donovani* strain, and used amastigote-harboring splenocytes to evaluate the antileishmanial activity of more than 4000 molecules. This medium-throughput screen revealed 84 small molecules with good antileishmanial activity and an acceptable toxicity evaluation [45]. Similarly, in a drug repurposing initiative, Fernandez-Prada et al. (2013) used BALB/c-derived splenic explants infected with *L. infantum* amastigotes expressing the infrared fluorescent protein IFP1.4 to evaluate the antileishmanial effect of anticancer-drug camptothecin and several analogues [37]. Markedly, and despite their many advantages, engineered parasites are not flawless, and different mitigation strategies should be taken into account in order to avoid any compensatory change in parasite metabolism or virulence (e.g., prioritize the use of integrative strategies to generate the strain) [35]. A final important remark is that, as has been recently demonstrated, there could be different compound efficiencies linked to the drug susceptibility background of the *Leishmania* strains used in the screening process (especially in the case of antimony susceptibility), which shows the potential value of including clinical isolates (and resistant strains) in the drug discovery cascade [47].
